Fig. 4: YidC mutated in the cytoplasmic region or hydrophilic groove binds Pf3 with different forces.
From: Monitoring the binding and insertion of a single transmembrane protein by an insertase
a, b Pf3 insertion into mutant ΔCH2 YidC (grey) or mutant R366E YidC (orange) and wt (purple) YidC proteoliposomes as measured by FCS. The Atto520 dye attached to the N-terminal end of Pf3 is quenched outside proteoliposomes and bursts fluorescence upon translocation via YidC into proteoliposomes. Data points represent means from 35 measurements and error bars sd. c, d (Un-)binding forces of ΔCH2 YidC (grey) or R366E YidC (orange) and the Pf3 polypeptide as detected by FD-AFM at different contact times (grey). Overlaid are (un-)binding forces of wt YidC and Pf3 (purple). Grey dots show data points from which the probability density functions (lines and shaded areas) were constructed. For better display data points were randomly scattered along the y-axis. Probability density functions from wt YidC were taken from Fig. 2c. Force–distance curves showing single unbinding events from either wt YidC, mutant ∆CH2 YidC or mutant R366E YidC are exemplified in Supplementary Fig. 16. e Multivariate linear regression (dashed lines) of wt (purple), R366E (orange) and ΔCH2 (grey) YidC built on the mean (un-)binding forces (dashed lines) of Pf3. The y-intercept equals 27.9 ± 2.2 pN (±95% CI), 32.5 ± 2.9 pN, and 26.5 ± 3.3 pN for wt, ΔCH2, and R366E YidC, respectively. Slopes of the (un-)binding forces equal 4.94 ± 0.91 pN s–1, 1.74 ± 1.48 pN s–1 and 1.66 ± 1.61 pN s–1 for wt, ΔCH2 and R366E YidC, respectively. Circular, triangle and square data points give means (wt YidC, nevents = 899; mutant R366E YidC, nevents = 408; mutant ΔCH2 YidC, nevents = 556) for each contact time. Shaded areas indicate 95% CI and error bars represent 95% CI of the means. Force distributions were statistically compared with a two-sided Mann–Whitney U-test showing P-values for each compared condition. Mean (un-)binding forces are summarized in Supplementary Table 1. Source data are provided as a Source Data file.
